The present disclosure relates, in various exemplary embodiments, generally to compositions and methods for enabling adhesion, proliferation and self-renewal maintenance of pluripotent, or undifferentiated, stem cells in vitro. These stem cells include embryonic stem cells, such as murine or human, and bone marrow stem cells.
A stem cell is an undifferentiated cell from which specialized cells are subsequently derived. Embryonic stem cells possess extensive self-renewal capacity and pluripotency with the potential to differentiate into cells of all three germ layers. They are useful for therapeutic purposes and may provide unlimited sources of cells for tissue replacement therapies, drug screening, functional genomics and proteomics. (Skottman, H., Dilber, M. S., and Hovatta, O. (2006); The derivation of clinical-grade human embryonic stem cell lines; FEBS Lett 580, 2875-2878).
Murine pluripotent embryonic cells can be maintained in a pluripotent state in in vitro cell culture conditions in the presence of Leukemia Inhibitory Factor (LIF). (Williams R L, Hilton D J, Pease S, Willson T A, Stewart C L, Gearing D P, Wagner E F, Metcalf D, Nicola N A, Gough N M (1988); Myeloid leukemia inhibitory factor maintains the developmental potential of embryonic stem cells, Nature 1988 Dec. 15; 336(6200):684-7).
Additionally, murine pluripotent embryonic cells can be maintained in a pluripotent state in vitro when cultured on mouse embryonic fibroblasts as feeder cells. Human embryonic cells also require feeder cells for maintenance in a pluripotent state in vitro or differentiation inhibitors like Noggin and/or high doses of basic fibroblast growth factor (FGF) when cultured on Matrigel™ (see for review: Skottman, H., Dilber, M. S., and Hovatta, O. (2006); The derivation of clinical-grade human embryonic stem cell lines; FEBS Lett 580, 2875-2878). However, the use of feeder cells has a number of drawbacks. For example, feeder cells can contain pathogens, such as viruses that can infect the stem cells (Hovatta, O., and Skottman, H. (2005); Feeder-free derivation of human embryonic stem-cell lines; Lancet 365, 1601-1603; Skottman, H., Dilber, M. S., and Hovatta, O. (2006); The derivation of clinical-grade human embryonic stem cell lines; FEBS Lett 580, 2875-2878).
Feeder-free systems that support human embryonic stem cell self-renewal require either i) Matrigel™ (Richards, M., Fong, C. Y., Chan, W. K., Wong, P. C., and Bongso, A. (2002); Human feeders support prolonged undifferentiated growth of human inner cell masses and embryonic stem cells; Nat Biotechnol 20, 933-936); (Xu, C., Inokuma, M. S., Denham, J., Golds, K., Kundu, P., Gold, J. D., and Carpenter, M. K. (2001); Feeder-free growth of undifferentiated human embryonic stem cells; Nat Biotechnol 19, 971-974); (Xu, R. H., Peck, R. M., Li, D. S., Feng, X., Ludwig, T., and Thomson, J. A. (2005); Basic FGF and suppression of BMP signaling sustain undifferentiated proliferation of human ES cells; Nat Methods 2, 185-190); or, ii) mouse feeders-derived extracellular matrix (Klimanskaya, I., Chung, Y., Meisner, L.; Johnson, J., West, M. D., and Lanza, R. (2005); Human embryonic stem cells derived without feeder cells; Lancet 365, 1636-1641) as adhesive substrata. However, these coatings are of xenogenic origin and therefore cannot be used in clinics according to FDA requirements (Hovatta, O., and Skottman, H. (2005); Feeder-free derivation of human embryonic stem-cell lines; Lancet 365, 1601-1603). These coatings also fail to fulfill criteria of defined system and non-immunogenicity, importance of which is discussed in (Hovatta, O., and Skottman, H. (2005); Feeder-free derivation of human embryonic stem-cell lines; Lancet 365, 1601-1603; Skottman, H., Dilber, M. S., and Hovatta, O. (2006); The derivation of clinical-grade human embryonic stem cell lines; FEBS Lett 580, 2875-2878).
During mammalian embryonic development, a fertilized oocyte first divides into two cells, followed by another cell duplication to generate a four-cell embryo. At the four-cell stage, the embryonic cells are bound together with the help of cell membrane proteins and also the molecules of a new connective tissue (extracellular matrix). The first extracellular matrix molecules to appear are basement membrane proteins, such as laminin and proteoglycan (Cooper, A. R., and MacQueen, H. A. (1983); Subunits of laminin are differentially synthesized in mouse eggs and early embryos; Dev Biol 96, 467-471) (Dziadek, M., and Timpl, R. (1985); Expression of nidogen and laminin in basement membranes during mouse embryogenesis and in teratocarcinoma cells; Dev Biol 111, 372-382). Subsequently, the embryonic cells start to differentiate into the three germ cell layers; ectoderm, endoderm and mesoderm, with initiation of morphogenesis. The extracellular matrix molecules, such as laminins are responsible for interactions with cell surface receptors, thus regulating cell behavior such as adhesion, proliferation, migration and differentiation (Colognato, H., and Yurchenco, P. D. (2000); Form and function: the laminin family of heterotrimers; Dev Dyn 218, 213-234), while other extracellular matrix components such as collagens of types I, II, III or IV primarily serve a mechanical supportive function (Aumailley, M., and Gayraud, B. (1998); Structure and biological activity of the extracellular matrix; J Mol Med 76, 253-265).
Extracellular matrix derived from murine fibroblasts, in combination with soluble differentiation inhibitors may be an adequate replacement for feeder cells (Klimanskaya, I., Chung, Y., Meisner, L., Johnson, J., West, M. D., and Lanza, R. (2005); Human embryonic stem cells derived without feeder cells; Lancet 365, 1636-1641), which demonstrates the critical role of extracellular matrix molecules. Laminins are large trimeric extracellular matrix proteins that are composed of alpha, beta, and gamma chains. There exist five different alpha chains, three beta chains and three gamma chains that in mouse and human tissues have been found in at least fifteen different combinations (Colognato, H., and Yurchenco, P. D. (2000); Form and function: the laminin family of heterotrimers; Dev Dyn 218, 213-234); (Aumailley, M., Bruckner-Tuderman, L., Carter, W. G., Deutzmann, R., Edgar, D., Ekblom, P., Engel, J., Engvall, E., Hohenester, E., Jones, J. C., et al. (2005); A simplified laminin nomenclature; Matrix Biol 24, 326-332). These molecules are termed laminin-1 to laminin-15 based on their historical discovery, but an alternative nomenclature describes the isoforms based on their chain composition, e.g. laminin-111 (laminin-1) that contains alpha-1, beta-1 and gamma-1 chains (laminin nomenclature: (Aumailley, M., Bruckner-Tuderman, L., Carter, W. G., Deutzmann, R., Edgar, D., Ekblom, P., Engel, J., Engvall, E., Hohenester, E., Jones, J. C., et al. (2005); A simplified laminin nomenclature; Matrix Biol 24, 326-332)).
Notwithstanding the above, there continues to be a need for providing compositions and methods for culturing and growing embryonic stem cells. In this regard, providing compositions and methods for enabling the proliferation and survival of pluripotent stem cells in vitro without use of differentiation inhibitory agents such as LIF or feeder cells would be advantageous.